Cholangiopathies are chronic, progressive diseases of the biliary tree, and can be either acquired or genetic. Regardless of etiology, cholangiopathies share common pathologic mechanisms, including inflammation, aberrant ductular proliferation, fibrosis, ductopenia, and cholestasis, which can over time result in tumorigenesis, cirrhosis, or liver failure. Despite recent advances in our understanding and diagnosis of these diseases, there are no proven therapeutic treatments for the majority of cholangiopathies. Thus, mechanistic-based studies that emphasize therapeutic development are desperately needed. Previous work has identified the Wnt/?-catenin signaling pathway as a modulatable target in mouse models of biliary injury such as 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet. Overexpression of a mutated non-degradable form of ?-catenin in transgenic (TG) mice subjected to long-term DDC results in a significant reduction in serum alkaline phosphatase, a common prognostic marker for biliary injury, and a concurrent increase in bile flow. Notably, this improvement was associated with widespread expression of biliary marker A6 in the hepatocytes (HC) of these TG mice. Further analysis revealed that TG had increased expression of biliary markers in HC as early as 1 month after DDC. During biliary injury, HC are known to alter their phenotype and acquire cholangiocyte (CC)-like features, a process known as cellular reprogramming. HC reprogramming may contribute to biliary repair by modifying bile to reduce toxicity, serving as a source of de novo CC to repair the biliary epithelium, or creating new channels to facilitate bile flow. Thus, the overarching hypothesis of the proposal is that activation of Wnt/?-catenin signaling in HC during cholestasis will induce reprogramming to a CC-like phenotype, and that this process will aid in restoring bile flow and reducing the severity of cholestatic liver disease. In aim 1, we will unambiguously determine if ?-catenin-overexpressing HC fully differentiate into functional CC or maintain an intermediate phenotype during cholestasis by isolating permanently labeled HC and their progeny and analyzing them through phenotypic characterization, functional tests, and transcriptomic analysis. In aim 2, we will characterize the mechanism by which Wnt/?-catenin activates a biliary phenotype in HC by using unbiased methods to identify the transcription factors downstream of ?-catenin in cholestasis, as well as evaluating the contribution of Yap signaling as a potential downstream effector of ?-catenin using an in vivo two-gene reporter system. In aim 3, we will determine whether activating ?-catenin in HC will enhance transdifferentiation into fully- functional CC in the absence of a functional biliary system. First, we will determine the effect of inhibiting or overexpressing ?-catenin on the formation of biliary structures in vitro (HC-derived organoid cultures). Next, we will exogenously activate ?-catenin using a Wnt agonist and assess its efficacy in alleviating cholestatic injury in mice with biliary insufficiency. Finally, we will utilize an immune-deficient, bile duct deficient model of liver repopulation and determine if transplanted TG HC have an advantage over wild-type HC in rescuing the phenotype. Thus, the proposed studies will further our understanding of the role of ?-catenin in HC reprogramming and biliary repair, and will provide highly significant information for therapeutic and translational use.